Automated solid phase protein synthesis using SEA peptide chemistry – SEA SPPS

Today, therapeutic proteins or proteins for R&D studies are mainly produced using recombinant techniques. Protein total chemical synthesis is emerging as an interesting complement and potential alternative to the use of living systems for producing small proteins. Importantly, chemical synthesis allows introductions of various and multiple modifications that are difficult, or even impossible to date, to set up by recombinant means. Chemical synthesis thus offers a great potential for tuning the properties of proteins such as potency, specificity, stability, immunogenicity, or the introduction of labels for specific detection purposes.
Conventional automated solid-phase peptide synthesis (SPPS) is used intensively for the production of R&D peptides and peptide therapeutics of moderate size, typically composed of less than 40 amino acids. Comparatively, synthesis of long peptides or small proteins (> 50 Aa) is still an industrial challenge.
Large polypeptides are best synthesized by native peptide ligation of unprotected and purified short peptide segments. While sequential native peptide ligations in solution enable protein synthesis on a mg scale, their actual limitations such as the difficulty to synthesize individual functionalized peptide segments and the lack of mature solid phase approaches and automated procedures have hampered their widespread application in academic labs and industrial units. A simple and efficient solid phase approach enabling the sequential assembly of individual peptide segments will be a significant breakthrough in the field, in a similar manner as SPPS revolutionized the field of peptide synthesis and initiated an important market.
Recently, a novel native peptide ligation method called bis(2-sulfanylethyl)amido (SEA) ligation was patented and disclosed by IBL in Lille (see the wikipedia web page SEA Native Peptide Ligation). This reaction and extended methodologies constitute a significant breakthrough in the field of protein total synthesis. In particular, SEA ligation, SEA peptide segment synthetic possibilities and the unique redox properties of SEA group can be combined for synthesizing proteins in the N-to-C direction as illustrated recently by our paper (Ollivier, N. et al. Angewandte Chemie Int. Ed. 2012, 51 (1): 209–213). This chemistry, when combined with linker technologies developed at CBM by the Synthetic Protein Chemistry team, is a potential solution to the solid phase assembly of proteins in the N-to-C direction, allowing a non-chromatographic purification of all the peptide fragments. Most peptide fragments arising from Fmoc-SPPS are only contaminated by N-terminal N-acetylated truncated co-products which do not interfere neither with the grafting of the first fragment nor the subsequent SEA ligations, and can therefore be eliminated through a simple wash of the solid-support after each step.
The goal of this SEA SPPS project is to develop, automate and validate linker technologies and SEA-based solid phase native peptide ligation protocols enabling the sequential assembly of peptide segments. We target the assembly of up to 6 segments and the total synthesis of a 170 amino acids long proteins.
Thanks to this proof of concept of the total synthesis of a medium sized protein using SEA SPPS, the technology will be ready for a transfert to a newly created start-up company focussed on the production of proteins for R&D, and licence contracting for use of SEA SPPS for the production of proteins in GMP environnement.